Listeria monocytogenes is a gram-positive, rapidly growing food-borne human pathogen which has been used extensively as a model facultative intracellular pathogen both in mice and tissue culture cells. The cell biology of intracellular growth can be divided into two broad stages; life in a vacuole and life in the host cytoplasm. The overall goal of the proposed research is to define the specific roles and regulation of gene-products which are required for the successful passage of L. monocytogenes through the vacuolar environment. The roles of four gene-products will be specifically evaluated; 1) Listeriolysin O (LLO), encoded by hly, a pore-forming hemolysin previously shown to be an essential determinant of pathogenicity necessary for lysis of the host vacuole; 2) PI-PLC, encoded by plcA, a phosphatidylinositol specific phospholipase C; 3) PC-PLC, encoded by plcB, a broad spectrum phospholipase C which hydrolyzes phosphatidylcholine; and 4) PrfA, encoded by prfA, a positive transcriptional activator of hly, plcA, and plcB. In Aim I, two hemolysins related to LLO (streptolysin O and perfringolysin O) will be evaluated to determine their capacity to complement a hly::Tn9l7 mutation. Characterization of these strains in animals and in tissue culture should provide insight into the specialized features of LLO which allow it to lyse the vacuole without damaging the host cell. In Aim II, the roles of PI-PLC and PC-PLC will be fully evaluated. In each case, in-frame deletion mutations will be introduced into the L. monocytogenes chromosome by homologous recombination and allelic exchange. Strains deleted for one or both genes will be constructed and evaluated by quantitative electron microscopy for their ability to lyse the host vacuole in both primary macrophages and cell lines. In Aim III, the role of the two prfA promoters in mediating vacuolar gene expression will be explored by constructing mutants lacking either or both promoters. hly transcription will be monitored using a hly::Tn9l7-lac gene fusion. In Aim IV, other genes required for the escape of L. monocytogenes from a vacuole will be isolated using intracellular methicillin selection.